This is the first revision of the R21 application, AI064118-01, which is in response to PA-03-080 Biodefense and Emerging Infectious Disease Research Opportunities. Enterocytozoon bieneusi is a clinically significant human enteric pathogen associated with immunodeficiencies including AIDS, and against which therapy is not readily available. Given the technical difficulties associated with the lack of in vitro and in vivo propagation methods and limited sources of spores, research on this organism has been limited. Thus, we propose to undertake an E. bieneusi genome sequence survey project which will generate several important genome-related resources (sequence, clones, libraries), which will be available to the research community. The specific aims are as follows: 1. Construct a small insert plasmid library from which approximately 40,000 clones will be sequenced. Sequence and assembly of reads to obtain 95-98% genome coverage, which should identify the majority of genes. A fosmid library will also be constructed and a 15-fold coverage will be performed. Master clone banks of both libraries will be created. 2. Perform functional genomic analyses and annotation of the generated sequence data. a. Using available computational tools, open reading frames, non-coding sequences, introns and regulatory elements will be identified. BLAST analyses will be performed to identify genes encoding proteins with high similarity to known proteins for assignment of function. b. A comparative analysis of the E. bieneusi genomic sequence with that of the Encephalitozoon cuniculi genome sequence will be carried out. Phylogenetic analysis of the cryptic mitochondrion and its function,and the ATP transporter genes will be undertaken. c. Establishment of a website to provide access to the sequence data and clones by the scientific community.